Projets de recherche

Code: 21A05XT

The design and synthesis of new anabolic androgenic steroids, characterized by reduced side effects and better oral bioavailability, begun shortly after the structure of testosterone was identified in 1935. Since then, the discovery of new steroids has been accompanied by studies on their metabolism, to understand how they could be metabolized by phase I and II enzyme isoforms that alter their toxicity, action, and elimination. The presence of pharmacologically active metabolites, their variable formation, and their potential interaction with other drugs and/or nutritional supplements, may indeed interfere with the analytical results of doping controls. Furthermore, although metabolite detection allows identification of the metabolic pathways involved in drug absorption, molecules with related structures and similar physicochemical properties may follow generally common metabolic pathways, but different biotransformation steps, making more challenging the selective and unambiguous identification of the original compound. The process of detecting, characterizing, and confirming metabolites is time-consuming and resource-intensive. The analytical strategy currently used to identify drug metabolites involves several steps, some of which are more influenced by knowledge bias. The combination of knowledge-based and machine-learning approaches represents an innovative and effective way to predict, identify and characterize drug metabolites, not only in anti-doping but also in clinical or forensic fields. 

To this end, the proposed systematic analytical protocol, capable of combining targeted and untargeted analyses, full scan, MS/MS experiments, and previous knowledge, represents a strategic tool for the rapid detection of drug metabolites. The use of accurate mass measurements by high-resolution mass spectrometry (HRMS) and the complementarity of liquid and gas chromatography ensures a high degree of completeness to the results. The processing of the raw data by multivariate analysis allows to focus on the specific features of the condition, without performing any a priori bias.

Code: 21A03MT

Phenethylamine (PEA) is listed on WADA´s Prohibited List as a stimulant and therefore its administration is forbidden in competition. As the human body naturally produces PEA, its presence in urine samples alone is not an indication for its misuse. Already in 2015, a potential metabolite indicative for the administration of PEA ((2-(3-hydroxyphenyl)acetamide sulfate) has been reported, based on pilot study results conducted with two individuals. During a follow up study encompassing more volunteers and different administration protocols, other potential metabolites were identified. Both PEA and its metabolites show are large inter-individual variance, i.e. the urinary concentrations of these compounds can be substantially elevated even without any administration simply due to the individual´s metabolism. This phenomenon is well described for urinary steroids and their concentrations as demonstrated for testosterone. Here, the carbon isotope ratios (CIR) are used to confirm the exogenous origin of elevated concentrations if found in urine samples of athletes. Similarly, within this research project a method suitable to determine Page 2/8 the CIR of PEA and its metabolites will be developed and validated according to WADA guidelines. Besides the development of a sophisticated sample clean-up procedure possible derivatization techniques will be investigated to ensure valid results. Finally, a reference population will be investigated to figure out the expected range of isotopic ratios for PEA and its metabolites. This will enable a clear differentiation between endogenous (produced naturally inside the human body) and exogenous (administered) PEA found in urine specimens.

Main Findings

Phenethylamine (PEA) is itemized as a stimulant on the Prohibited List of the World Anti-Doping Agency since 2015. It represents a naturally occurring trace amine and has been investigated in the 1970s and 80s regarding its medical potential as a modulator in the central nervous system. Today it is sold as a dietary supplement, marketed for its mood enhancing effects and as a potential treatment for weight loss.

Its detection in sports drug testing remains complicated as PEA is an endogenous substance, i.e. it is present in urine, at least in trace amounts. Investigations into a reference population demonstrated that the found urinary concentrations show a large inter-individual variation. Interestingly, after the oral administration of PEA, its urinary concentrations were found only slightly elevated. Establishing a urinary concentration threshold for PEA was therefore not successful and further studies focused on potential metabolites of PEA identifying one sulphated minor metabolite and phenylacetylglutamine (PAG) as the most abundant metabolite of PEA in urine. Again, the biological variability of these urinary metabolites complicated establishing a threshold and only the combination of both parameters in a binary logistic regression enabled to identify post-administration samples.

Aim of this research was the development and validation of an isotope ratio mass spectrometry-based method for PEA and PAG to differentiate between the endogenous or exogenous source of these metabolites considering their carbon isotope ratio (CIR). Especially for PAG, the developed method met all expectations and reference population-based thresholds were established. Unfortunately, both endogenous PEA and PAG showed unexpectedly depleted CIR and the found difference between exogenous and endogenous PEA was only around 3 ‰. According to these relatively similar CIR, the opportunities for detecting an oral administration of PEA were very short, and after a single oral dose none of the volunteers was found with CIR outside the population-based thresholds.

In athletes with relatively enriched CIR the developed method will most probably not result in an adverse finding as here the impact of the exogenous PEA will not influence the determined CIR of urinary PAG beyond the established threshold.

Further investigations will be necessary to elucidate the potential of IRMS to detect the misuse of PEA, e.g. by means of stable isotopes other than carbon.

Code: 21A02MP

In human sports the application of glucocorticoids is limited by anti-doping regulations, however, recent statements of professional cyclists underline the high potential of misuse. Currently, for glucocorticoids a reporting level of 30 ng/mL in urine is established in doping control analysis. Etamivan is classified as respiratory stimulant, that is also prohibited in sports. Due to its high metabolic sulfonation in humans, in recent external quality assessment schemes (EQUAS) etamivan-sulfate was explicitly integrated as target analyte besides the parent compound etamivan. At present a reporting limit of 50 ng/mL for stimulants is specified. Both classes are highly prone to matrix interferences in LC-ESI-MS/MS that may confound quantitation and thus influence decision of reporting. As orthogonal technique, SFC-MS/MS may help to overcome these issues. Thus, the project aims to investigate its potential for determination of glucocorticosteroids and etamivan and its sulfoconjugate. Furthermore, the LC- and SFC-based methods will be compared in terms of matrix interferences and accuracy of determination.

Code: 21A04RM

In the glucuronated fraction, the differences among Δδ13C pairs are known to reflect the metabolic isotopic fractionation and potential method bias. This is known as well, to be magnified in the sulfate fraction. After the publication of some results concerning the sulfate fraction analysis advantages, WADA TD2021IRMS states that Epiandrosterone could be used as an additional TC for GC-C-IRMS
analysis to determine the administration of testosterone or its precursors and the decision rule for IRMS positive test is based on the absolute Δδ13C value of ERC-EpiA pair higher than 4‰. No
specification is explicitly done about which ERC should be used to evaluate an analyte from the sulfate fraction and in which fraction the ERC should be present.

The project aims to study the differences between the δ13C and Δδ13C values obtained taking into consideration the ERC in glucuronated and sulfate fraction of urine, during the evaluation of
sulfated target compounds. It will be studied the classic ERCs: Pregnanediol, Pregnanetriol, 11β-hydroxy-androsterone,11β-hydroxy-etiocholanolone and Androstenol. Sulfoconjugation of these compounds is not extensively published but we do not expect more than 10% based on the structure. In this case, a preliminary and alternative study of estrogens will be carrying out as well as androst-5-ene-3β,17α-diol proposed previously.

If the goals of the proposed project are reached, we should be able to align the results based on ERC and TC both excreted as sulfo-conjugates. Besides, the bias should be minimized because the
already described difference between CIR of glucuronate and sulfate fractions could be avoided. Finally, we expected to increase the knowledge regarding the δ13C of ERCs and Δδ13C pairs, all in
sulfate fraction from the Reference Population Data (at least for males) which can contribute to the selection of a sensitive and/or affordable ERC for IRMS confirmation in sulfate fraction of the urine.

Dr. Nikola Jojic, University Business Academy Novi Sad, Faculty of Pharmacy Novi Sad

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